Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.
In polycystic kidney disease (PKD), Bardet-Biedl Syndrome (BBS), and other disorders, mutations in cilia-associated structural or signaling proteins cause insensitivity to external mechanical and diffusible signaling cues, resulting in disorganized, hyperplastic cell growth (Benzing and Walz, 2006; Pan et al., 2005; Singla and Reiter, 2006). On the organismal level, ciliary defects produce renal cysts, infertility, respiratory disorders, situs inversus, and predisposition to obesity, diabetes, and hypertension. Notably, recent studies have shown that the Hedgehog, Wnt, PDGFαα, and other signaling cascades are coordinated at cilia (Cano et al., 2004; Huangfu and Anderson, 2005; Liu et al., 2005; Schneider et al., 2005; Simons et al., 2005; Tanaka et al., 2005). The frequent deregulation of these pathways during cell transformation, together with the common disappearance of cilia in transformed cells, raises the possibility that defective ciliary signaling may promote cancer.
Although an increasing number of proteins are being defined as ciliary structural components or cilia-associated signaling proteins, very little is currently known about the cellular machinery controlling the formation and resorption of cilia. It has long been known that cilia are regulated dynamically throughout the cell cycle. In many cells, resorption occurs at mitotic entry, and reappearance after progression into G1. However, resorption is not solely linked to mitotic entry, with some cells undergoing waves of resorption at different points in cell cycle: for example, Tucker et al. have noted ciliary resorption as cells emerge from quiescence, prior to S-phase (Quarmby and Parker, 2005; Rieder et al., 1979; Tucker et al., 1979). Given their increasingly apparent role in detecting and transmitting extracellular signals, regulated formation, disassembly, or shortening of cilia may play an important role in cellular growth controls, serving as a rheostat to limit response to overly persistent or abnormal cell growth cues in the extracellular environment.
A cilium arises from a basal body, a structure that differentiates from one of the centrioles in the centrosome in non-proliferating cells and organizes the microtubule bundles that constitute the ciliary axoneme. Cilia are evolutionarily related to the motile flagella of lower eukaryotes, such as the green algae Chlamydomonas. Genetic studies in Chlamydomonas have recently begun to dissect the process of flagellar resorption (Bradley and Quarmby, 2005; Marshall et al., 2005; Pan and Snell, 2005; Quarmby, 2004). These studies have identified altered functionality of the intraflagellar transport (IFT) machinery and destabilization of the axoneme as hallmarks of disassembly, and implicated CALK and other kinases as regulators of disassembly. The means by which CALK becomes activated at initiation of disassembly and the critical CALK, effectors in the disassembly process remain unknown, as does the relevance of these observations to higher eukaryotes.
CALK is very distantly related to the human Aurora A (AurA) kinase, with 55% similarity centered on the protein catalytic domain. In humans, Aurora A (AurA) is a centrosomal kinase that regulates mitotic entry through activation of Cdk1-cyclin B and other substrates that organize the mitotic spindle (Bischoff et al., 1998; Marumoto et al., 2005). AurA amplification or activation is common in many cancers characterized by centrosomal amplification and genomic instability (Anand et al., 2003; Goepfert et al., 2002; Gritsko et al., 2003). In the past year, altered expression of the HEF1 (Law et al., 1996; O'Neill et al., 2000) scaffolding protein has recently been identified as part of a pro-metastatic signature in breast cancer (Minn et al., 2005), shown to contribute to the aggressiveness of glioblastomas (Natarajan et al., 2006), and found to be critical for progression to metastasis in melanomas (Kim et al., 2006). HEF1 is best known as a transducer of integrin-initiated attachment, migration, and anti-apoptotic signals at focal adhesions (O'Neill et al., 2000).